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Journal of Virology, June 2005, p. 6859-6867, Vol. 79, No. 11
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.11.6859-6867.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Effect of Polypurine Tract (PPT) Mutations on Human Immunodeficiency Virus Type 1 Replication: a Virus with a Completely Randomized PPT Retains Low Infectivity

Lesa R. Miles,1 Beth E. Agresta,2 Mahfuz B. Khan,1 Shixing Tang,2,{dagger} Judith G. Levin,2 and Michael D. Powell1*

Department of Microbiology, Biochemistry and Immunology, Morehouse School of Medicine, Atlanta, Georgia 30310,1 Laboratory of Molecular Genetics, National Institute of Child Health and Human Development, Bethesda, Maryland 208922

Received 14 October 2004/ Accepted 20 January 2005

We introduced polypurine tract (PPT) mutations, which we had previously tested in an in vitro assay, into the viral clone NL4-3KFS{Delta}nef. Each mutant was tested for single-round infectivity and virion production. All of the PPT mutations had an effect on replication; however, mutation of the 5' end appeared to have less of an effect on infectivity than mutation of the 3' end of the PPT sequence. Curiously, a mutation in which the entire PPT sequence was randomized (PPTSUB) retained 12% of the infectivity of the wild type (WT) in a multinuclear activation of galactosidase indicator assay. Supernatants from these infections contained viral particles, as evidenced by the presence of p24 antigen. Two-long terminal repeat (2-LTR) circle junction analysis following PPTSUB infection revealed that the mutant could form a high percentage of normal junctions. Quantification of the 2-LTR circles using real-time PCR revealed that number of 2-LTR circles from cells infected with the PPTSUB mutant was 3.5 logs greater than 2-LTR circles from cells infected with WT virus. To determine whether the progeny virions from a PPTSUB infection could undergo further rounds of replication, we introduced the PPTSUB mutation into a replication-competent virus. Our results show that the mutant virus is able to replicate and that the infectivity of the progeny virions increases with each passage, quickly reverting to a WT PPT sequence. Together, these experiments confirm that the 3' end of the PPT is important for plus-strand priming and that a virus that completely lacks a PPT can replicate at a low level.

* Corresponding author. Mailing address: Department of Microbiology, Biochemistry and Immunology, Morehouse School of Medicine, 720 Westview Dr. S. W., Atlanta, GA 30310. Phone: (404) 752-1582. Fax: (404) 752-1179. E-mail: powellm{at}msm.edu.

{dagger} Present address: Laboratory of Molecular Virology, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, MD 20892.

Journal of Virology, June 2005, p. 6859-6867, Vol. 79, No. 11
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.11.6859-6867.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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